Living organisms have natural immune system that quickly recognizes and eliminates pathogens such as virus, bacterium and the like that entered the living body. In the living organisms invaded by pathogens, airway epithelial cells, dendritic cells and the like multilaterally recognize partial structure of pathogens via natural immunoreceptors such as Toll-like receptor, RIG-I-like receptor and the like, activate natural immunity and cause a natural immune response such as production of Type I interferon (IFN) and inflammatory cytokines. The constituent components of pathogens recognized by natural immunity and substances mimicing same are called Immune Potentiators, and applied researches thereof as an adjuvant for pharmaceutical products or vaccines are ongoing. Particularly, in the use for infection vaccines and cancer vaccines, its practicalization as an adjuvant capable of improving basic property while securing safety is expected. One of such immune potentiators is artificially synthesized double-stranded ribonucleic acid (dsRNA).
Representative examples of the artificially synthesized dsRNA include poly(A:U) wherein the single-stranded ribonucleic acids (ssRNAs) constituting a double-strand are adenylic acid homopolymer and uridylic acid homopolymer, poly(G:C) wherein ssRNAs constituting a double-strand are guanylic acid homopolymer and cytidylic acid homopolymer, polyIC wherein ssRNAs constituting a double-strand are inosinic acid homopolymer and cytidylic acid homopolymer, poly(I:C12U) wherein ssRNAs constituting a double-strand are polymer C12U of an about 12:1 mixture of cytidylic acid and uridylic acid and inosinic acid homopolymer, and the like. These homo dsRNAs, particularly polyIC and poly(I:C12U), are candidates for a therapeutic drug for viral diseases and anticancer drugs, and a number of basic researches and application studies have been conducted. Among them are reports on the observation of side reaction in non-clinical tests, and such finding is one of the matters of concern that prevent practicalization.
When the efficacy and side reaction of dsRNA such as polyIC and the like are discussed, the relationship with the chain length should be noted. A discussion with the knowledge of a finding that polyIC having a chain length of several kbp or more shows stronger toxicity than short chain polyIC is necessary.
The chain length as used in the present invention means the same as the base number of RNA. The unit of the chain length of ssRNA is expressed in base or kilobase (1 kb=1000 bases), and that of dsRNA is expressed in base pairs (bp) or kilobase pairs (1 kbp=1000 bp).
The weight average chain length of ssRNA and dsRNA is preferably a chain length determined by a gel permeation chromatography (GPC) analysis method. To be specific, GPC analysis is performed using dsRNA or dsDNA having a known molecular weight as a standard product, and average chain length and median value of the chain length are calculated from the obtained data. Another method includes determining sedimentation coefficient S (20,w) by a density gradient sedimentation velocity method, and estimating the chain length of ssRNA and dsRNA from an experimental formula (The Journal of Biochemistry (1961)50:377).
Non-patent document 1 discusses the relationship between the chain length of polyIC and median lethal dose (LD50) in mouse intraperitoneal administration and describes that LD50 of polyIC decreases as the chain length becomes longer from 0.1 kbp to 6 kbp, namely, that the toxicity becomes stronger. To be specific, LD50 of polyIC having a centrifugation sedimentation coefficient 11.6 S (weight average chain length 2 kbp) is about ⅕ to that of polyIC having a centrifugation sedimentation coefficient 4.2 S (weight average chain length 0.1 kbp), and about ⅔ to that of polyIC having a centrifugation sedimentation coefficient 8.2 S (weight average chain length 0.8 kbp). Patent document 1 describes that a mouse intravenously administered with polyIC having a centrifugation sedimentation coefficient of not less than 13 S (i.e., weight average chain length of not less than 3 kbp) showed a 61% decrease in the bone marrow cell number, whereas a mouse intravenously administered with polyIC having a centrifugation sedimentation coefficient of 8 S (weight average chain length 0.75 kbp) did not show a decrease in the bone marrow cell number. Similarly, a mouse intravenously administered with poly(I:C12U) having a centrifugation sedimentation coefficient of not less than 13 S showed a 59% decrease in the bone marrow cell number, and a mouse intravenously administered with poly(I:C12U) having a centrifugation sedimentation coefficient of 9 S (weight average chain length 1.0 kbp) did not show a decrease in the bone marrow cell number.
On the other hand, non-patent document 1 describes that the strength of the Type I IFN inducing action of polyIC and the induction time are maintained more strongly and longer by polyIC having a longer chain length. Non-patent document 2 describes that chemically synthesized 70 bp polyIC shows RIG-1 binding ability in mouse fetal fibroblasts but the IFNβ induction activity markedly decreases as compared to 1.2 kbp polyIC. Non-patent document 3 describes that the chain length of dsRNA necessary for dsRNA dependent protein kinase reaction, which is one of the virus defense mechanisms that are activated by Type I IFN as a signal, and dsRNA dependent 2′,5′-oligoA synthetase reaction is not less than about 40-60 bp.
With such findings as background, the chain length of dsRNA that functions as an adjuvant and has low toxicity is generally considered to be preferably 0.1 kbp-2.0 kbp.
Artificial homo dsRNA is generally obtained by a production method including synthesizing ssRNA by using ribonucleotide diphosphate as a substrate and enzymes such as poly ribonucleotide nucleotidyl transferase and the like, and forming a double-strand by an annealing treatment. The enzymatically synthesized ssRNA is, from the property of the enzymatic reaction, a mixture with various chain lengths. In addition, ssRNA is physicochemically unstable and, in our experience, a phosphodiester bond is broken in a neutral aqueous solution under an atmosphere of not less than about 50° C., thus resulting in the division of polymer. Furthermore, when homopolymers such as inosinic acid polymer and cytidylic acid polymer are annealed, plural ssRNAs are successively linked depending on the annealing conditions, as a result of which the average chain length of the resulting dsRNA becomes long. From such technical background, dsRNA reagents commercially available at present often have different average chain lengths according to the makers and according to lots, and the toxicity thereof is also assumed to be different for each product lot.